1. Field of the Invention
The present invention relates to bio-analysis system and method, and particularly an improved system and method for carbohydrate analysis.
2. Description of Related Art
During the last two decades, chromatographic separation of complex carbohydrate mixtures has undergone a remarkable development. The earlier strategies involved the use of gas chromatography (GC) and GC/MS (Mass Spectroscopy) for quantification of monosaccharides and linkage determinations. To increase the volatility and hydrophobicity of sugar molecules, permethylation was utilized in the gas-phase analyses. While permethylation is still practiced to this date to enhance certain analytical objectives, low volatility of larger oligosaccharides made the GC approach somewhat unattractive. A search for optimum separation media in the liquid-phase separation of glycans has been evident for a number of years. Additionally, carbohydrates in their native state yield poor detection in chromatography, because of the lack of chromophores in their molecules. A derivatization (typically occurring at the reducing end of oligosaccharide chains) can introduce a chromophore or a fluorophore for the sake of detection and simultaneously enhance the solutes hydrophobicity for an appropriate separation.
A significant breakthrough in carbohydrate chromatographic analysis was achieved through combining anion-exchange chromatography of glycans with pulsed-amperometric detection (PAD). Under highly alkaline pH conditions, carbohydrates become charged, interactive with the ion-exchange resins, and detectable electrochemically. Following a series of incremental improvements, this form of chromatography was commercialized, becoming a standard tool of glycobiology. While this combination allows fairly effective separations of unmodified carbohydrates and quantification in the picomole range, further structural investigations of the separated glycans are complicated by their recovery from high-salt media. Carbohydrate analysis applied in laboratories also utilizes slab gel-based electrophoresis technologies. However, slab gel electrophoresis for carbohydrate analysis is labor-intensive, low throughput and low resolution. Traditional gel-based electrophoresis methods currently used for carbohydrate analysis take hours, if not days, to produce results with many cumbersome manual procedures, which are subject to human errors.
High-performance capillary electrophoresis (HPCE) now represents a set of powerful electromigration techniques whose impact has been felt in virtually all areas of biochemical analysis, including carbohydrate separations. HPCE is a micro fluidic approach to gel electrophoresis, whose greatest advantage is its diverse range of applications. CE technology is commonly accepted by the biotechnology industry, as a reliable, high resolution and highly sensitive detection tool.
While the first applications of HPCE to sugar analysis were described more than a decade ago, high interest HPCE methodologies remain to this date. Unprecedented separations of highly complex oligosaccharide mixtures were demonstrated, as was the resolution of sugar optical isomers and extremely sensitive detection of fluorescently labeled glycans from single biological cells through laser-induced fluorescence (LIF) detection. To comply with the sensitivity requirements, labeling with fluorophoric group is often the requirement for HPCE carbohydrate applications. CE with laser-induced fluorescence (LIF) is one of the most powerful analytical tools for rapid, high sensitivity and high-resolution bio-analysis type applications.
CE with laser-induced fluorescence (LIF) combined with derivatization agents, such as APTS, have been sought for detection of glycans. APTS was utilized for the labeling of N-glycans released from ribonuclease B and fetuin prior to their analysis by gel CE. It was also utilized in the analysis of N-glycans by others, including glycoproteins such as ribonuclease B, fetuin, recombinant human erythropoietin, karrikrein, and chimeric recombinant monoclonal antibody.
Recently, a complete method for analysis of N-glycans has been derived from glycoproteins. It is based on a combination of specific chemical and enzymatic conversions coupled with CE/LIF. N-Glycans are released enzymatically from glycoproteins and derivatized with APTS under mild reductive amination conditions to preserve sialic acid and fucose residues. The method successfully profiled the heavily sialylated N-glycans. A method for multistructure sequencing of N-glycans by gel CE and exoglycosidase digestions has also been devised.
The current CE systems with laser-induced fluorescence (LIF) detection mechanism that use multiple capillaries/channels for high-throughput applications are complicated in design and operation of the instrument. These systems utilize scanning optical detection mechanisms, which are much more bulky, sensitive in optical alignment and naturally more expensive than the traditional slab gel based systems. The expensive multi-capillary electrophoresis-based systems are thus out of reach for all but a few well-funded laboratories and seem to be a barrier for the expansion of the high-throughput carbohydrate analysis business.
It is therefore desirable to develop a simpler and much improved technology to provide lower per sample-cost, rapid and multi-channel type analysis with high efficiency, sensitivity, throughput, and ultimately, standardization for routine carbohydrate analysis.